Modulating the Cellular Uptake of Fluorescently Tagged Substrates of Prostate-Specific Antigen before and after...

A series of peptides based on the prostate-specific antigen (PSA)-specific sequence histidine-serine-serine-lysine-leucine-glutamine were functionalized with an anthraquinone fluorophore at the C-terminal residue side chain using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The effect of incorporating a negatively charged N-terminal tetra-glutamic acid group into the substrate and the effect of masking the negatively charged C-terminal carboxylic acid functionality of the substrate were investigated using confocal fluorescence microscopy in two cell lines, DLD-1 and LnCaP. The addition of a tetra-glutamic acid group to the N-terminus of the intact sequence was shown to reduce cellular uptake of the intact substrate prior to activation by PSA. In contrast, masking the C-terminal carboxylic acid group of the substrate as a methyl ester was shown to improve cellular uptake of the peptide fragment after activation by PSA. The synthesized C-terminal methyl ester substrates with the anthraquinone attached to the side chain were confirmed to be cleaved by PSA in LC-MS analysis, and the cytotoxicity of the substrates was shown to increase in the presence of PSA, consistent with cleavage and uptake of the C-terminal fragment. The results indicate that C- and N-terminal functionalization of peptide substrates targeting PSA can be used to modulate the cellular uptake of peptides before and after enzymatic activation, which may thus be an important consideration in the design of tumor-activated prodrugs.